TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

Upon the completion of incubation, the culture supernatant was removed and cells were washed three times in sterile phosphate-buffered saline (PBS) to remove any conidia that did not bind/attach to cells. Total RNA was prepared from infected and uninfected A549 cells with TRIzol (Sigma Chemical Co., St. Louis, MO) in the dish (1ml TRIzol per 10 cm2), respectively. The samples were stored at -80°C prior to RNA extraction. RNA extraction and RNA-Seq analysis were performed by BGI Tech company (Shenzhen, China). Briefly, after the total RNA extraction and DNase I treatment, the mRNA was fragmented into short fragments. Then cDNA was synthesized using the mRNA fragments as templates. After agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library. At last, the library could be sequenced using Illumina HiSeq 2000.

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Manufacturer protocol

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